Gluten Hydrolysate Research Articles

Increased stability and intracellular antioxidant activity of chlorogenic acid depend on its molecular interaction with wheat gluten hydrolysate

Gastrointestinal stability and cell entry efficiency affect the biological accessibility of chlorogenic acid (CGA). Here, wheat gluten hydrolysate (WGH) was proven to improve the stability of CGA during simulated gastrointestinal digestion, promote the intestinal epithelial cell entry efficiency of CGA, and increase its intracellular antioxidant activity. The interaction between WGH and CGA was studied by fluorescence quenching and molecular dynamics simulations. The thermodynamic parameters and molecular dynamics simulation analysis showed that the interaction between WGH and CGA was dependent on hydrogen bonding and hydrophobic and electrostatic interactions. Analyses of the binding sites of WGH showed that Arg12, Arg49, Lys54, and Pro74-Gln89 had strong interactions with CGA molecules. This interaction between CGA and WGH was related to both electrostatic interactions and their respective concentrations. Taken together, the stability, intestinal epithelial cell entry, and antioxidant activity of CGA can be increased by its molecular interactions with WGH.

Identification of novel angiotensin I-converting enzyme (ACE) inhibitory peptides from wheat gluten hydrolysate by the protease of Pseudomonas aeruginosa

Abstract The feasibility of a protease of Pseudomonas aeruginosa (PaproA) to prepare angiotensin I-converting enzyme (ACE) inhibitory peptides from wheat gluten was evaluated according to physiochemical and antihypertensive performances. The wheat gluten hydrolyzed by Alcalase plus PaproA (WGH_APae) to produce small fragments (

The potentials of probiotics on gluten hydrolysis; a review study.

Celiac disease (CD) is an autoimmune disorder of the small intestinal mucosa in genetically susceptible subjects consuming gluten. Gluten in wheat, rye and barley is harmful for some individuals and leads to various symptoms. Research has shown that treatment with probiotics in CD patients could improve the symptoms by the gluten hydrolysis. For this purpose, different databases such as Medline, PubMed, Scopus, and Google Scholar were searched using the following keywords: Celiac disease, Wheat flour, Gluten, glutamine, Probiotic, Bifidobacterium, Lactobacillus, Enzymes, Wheat allergy, Immune system, T cells, HLA-DQ2, HLA-DQ8, Gluten-free diet, Proteolysis, α2-gliadin fragment, Gliadin, 33-mer peptide, and Zonulin. The search aimed to retrieve the articles published during 2000-2019. Today, a gluten-free diet (GFD) is the only celiac disease treatment. Biotechnological strategy based on probiotic treatment could degrade gluten. Research has shown that combination of the probiotic enzyme is more effective than single probiotic on gluten hydrolysis. The result of different studies showed that probiotic mixture has the capacity to hydrolyze a considerable concentration of the 33-mer of gliadin completely. The present study was aimed to investigate associations between the capacities of probiotics on gluten hydrolysis.

Antibodies raised against a Sunn bug (Eurygaster integriceps Put.) recombinant protease, rGHP3p2, can inhibit gluten-hydrolyzing activity.

Sunn pest or Sunn bug, Eurygaster integriceps Put., salivary gland proteases are responsible for the deterioration of wheat flour quality during dough mixing, resulting from gluten hydrolysis. These proteases are highly heterogeneous and show low sensitivity to most types of proteinaceous inhibitors, meaning that such inhibitors cannot be used to prevent gluten damage. The present study describes the generation of a specific peptide antibody, raised against the active center of the recombinant gluten‐hydrolyzing protease (GHP3). The recombinant protein, encoding two repeats of the GHP3 sequence element involved in forming the S4 pocket and binding of substrate at position P4, was designed and expressed in Escherichia coli. The antibodies raised to this recombinant protein showed inhibitory activity against the GHP3 protease. The results indicate that it is possible to design specific antibodies to inhibit wheat‐bug gluten‐hydrolyzing proteases.

Evaluation of N-terminal labeling mass spectrometry for characterization of partially hydrolyzed gluten proteins

Gluten, a group of proteins found in wheat, barley, and rye, is the trigger of celiac disease, an immune disorder that affects about 1% of people worldwide. The toxicity of partially hydrolyzed gluten (PHG) in fermented products is less well understood due to the significant analytical challenges in PHG characterization. In this project, an N-terminal labeling mass spectrometry method, terminal amine isotopic labeling of substrates (TAILS), was optimized for the in-depth analysis of PHG and validated using a test protease (trypsin) with known cleavage specificity. Gluten N-termini in test and control groups were labeled with heavy and light formaldehyde, respectively. Trypsin-generated neo N-termini were identified by exhibiting an MS1 Log2 H:L peak area ratio with a significant difference (p < .01) from zero and native N-termini with no significant difference from zero (p > .01). Using this strategy, all abundant, theoretical, test protease-generated peptides in exemplar alpha/beta gliadins and gamma gliadins were identified. SignificanceThis study is the first study that modified and evaluated TAILS analysis for the analysis of partially hydrolyzed gluten proteins. The evaluation indicated that the TAILS analysis could be modified and expanded to the identification of multiple protease cleavage sites in gluten proteins and is worth further evaluation as a novel strategy for the analysis of natural hydrolysis of gluten in food processes. This strategy also may be further applied to characterize a broader range of partially hydrolyzed allergens in foods and provide reference for their safety assessment to both industry and regulatory authorities.

Presence of Exopeptidase-Resistant and Susceptible Peptides in a Bacterial Protease Digest of Corn Gluten.

Corn gluten hydrolysate (CGH) was prepared by food-grade bacterial proteases, alcalase and neutral protease. Digestion of CGH with carboxypeptidase A and leucine aminopeptidase extensively changed the elution patterns of peptides as observed from reversed phase high performance liquid chromatography-mass spectrometry (LC-MS), whereas digestion with pepsin and trypsin hardly affected the elution patterns. Twenty-five major peptides in CGH were identified. After digestion with exopeptidases, only prolyl dipeptides and pyroglutamyl di- and tripeptides remained, whereas the other 17 peptides completely disappeared. On the other hand, all 25 peptides remained after digestion with pepsin and trypsin. These facts suggest that a majority of short-chain peptides in food protein hydrolysates are degraded by exopeptidases during digestion and absorption processes. Thus, susceptibility to exopeptidases should be considered for prediction of bioactive peptide upon ingestion, which has not been considered in most of previous studies on food-derived bioactive peptides.

Prolyl endopeptidase-degraded low immunoreactive wheat flour attenuates immune responses in Caco-2 intestinal cells and gluten-sensitized BALB/c mice

Targeted degrading Aspergillus niger-derived prolyl endopeptidase (AN-PEP) is promising in gluten hydrolysis because it specifically cleaves the proline-rich sites in gluten. The current study aims to understand the safety aspects of AN-PEP hydrolysed low immunoreactive wheat flours by testing immune responses using cell line and animal models. In the AN-PEP hydrolysed wheat flour (AN-PEP HWF) gliadin extract, there was no increase in the levels of zonulin-1 (Zo-1) and pro-inflammatory cytokines (IL-6 and IL-8) but a significant increase was noted in the control wheat flour (CWF) gliadin-treated Caco-2 cells. The Zo-1 localization in Caco-2 cells was significantly noted in the reacted positive fluorescence cells that were treated with the control wheat flour. Further, a safety evaluation of HWF was carried out in gluten-sensitized BALB/c mice. Mouse anti-gliadin (IgG, IgA and IgE) antibodies were significantly generated in the CWF treated animals rather than the AN-PEP HWF groups. The serum pro-inflammatory (IL-1β, IL-4, IL-6, IL-15, TNF-α and IFN-γ) markers were observed in significant levels in CWF challenged mice and a similar trend was observed in ex-vivo splenocyte cells. A small intestine histopathological sectioning revealed that there are no abnormalities or structural changes in AN-PEP HWF challenged mice.

Lactobacillus plantarum ITM21B fermentation product and chickpea flour enhance the nutritional profile of salt reduced bakery products

The study aimed at improving the nutritional profile of yeast leavened salt reduced sliced bread and puccia type bread fortified with a wheat-based Lactobacillus plantarum ITM21B fermentation product (Bio21B). The protein content of bread made under laboratory conditions was increased by using: (i) chickpea flour (CF) (15% wt/wt flour) and Bio21B or (ii) the Bio21B containing a fungal protease to favour the gluten hydrolysis. Products showed increased protein and total amino acid content and improved protein digestibility. Moreover, the formula significantly affected the protein pattern of breads which, according to the results of the microfluidic two-dimensional electrophoresis (μ2DE) protein pattern, were discriminated as observed by the PCA plot. The use of CF was validated at industrial pilot plant producing salt reduced sliced bread and puccia type bread. The resulting products showed improved nutritional profile and a sensory quality comparable to the company’s products containing salt.

Efficient fermentation of very high-gravity worts by brewer's yeast with wheat gluten hydrolysates and their ultrafiltration fractions supplementations

Very high-gravity (VHG) worts (24 °P) supplemented with wheat gluten hydrolysates (WGH) and their ultrafiltration fractions (Mw > 1 kDa and <1 kDa fractions) were used to evaluate their effects on physiological activity and ethanol fermentation of brewer's yeast. The results demonstrated that WGH and their ultrafiltration fractions supplementations could obviously improve the biomass and viability of yeast, and further enhance the sugar utilization and ethanol production under VHG fermentation. Supplementation with Mw < 1 kDa fraction exhibited the maximum biomass (6.90 g/L dry cell), sugar utilization (79.79%) and ethanol concentration (11.88%, v/v) during VHG fermentation. Meanwhile, the addition of Mw < 1 kDa fraction significantly decreased the plasma membrane permeability and intracellular reactive oxygen species (ROS) accumulation, and increased the mitochondrial membrane potential in cells during VHG fermentation. Moreover, Mw < 1 kDa fraction supplementation caused the highest accumulations of intracellular trehalose and glycerol, which were 12.10% and 31.22% higher than the control, respectively. These results illustrated that WGH (especially Mw < 1 kDa fraction) addition was an economic and efficient method to improve physiological activity and ethanol fermentation of brewer's yeast in VHG worts.

Wheat Gluten and Its Hydrolysates. Possible Fields of Practical Use

Wheat gluten is a group of water-insoluble proteins of wheat endosperm. As a co-product of the starch and bioethanol production, gluten is available in fairly large quantities. Therefore, the search for optimal ways of its industrial utilization is an important economic task. The food industry is a traditional area for gluten application. However, the study of its structural and functional properties allowed expanding the area of the gluten use due to its various modifications, in particular, enzymatic hydrolysis. As compared to native gluten, the gluten hydrolysates have better foaming and emulsifying abilities, as well as higher water solubility. Biologically active properties of hydrolysates (antioxidant, hepatoprotective and angiotensin-converting enzyme inhibiting) were also shown. The search for and selection of proteolytic enzymes or their combinations, and optimization of the technological process will permit to obtain the hydrolysis products with the required characteristics, expand their use in industry and reduce the cost of their production.

Chemical characteristics and antioxidant properties of wheat gluten hydrolysates produced by single and sequential enzymatic hydrolyses using commercial proteases and their application in beverage system

The objective of the study was to determine chemical characteristics and antioxidant properties of wheat gluten hydrolysates (WGH) and utilize the WGH as ingredients in a beverage system. Single (2, 6, 12, and 24 h) and sequential enzymatic hydrolyses (8, 10, and 12 h) were conducted using commercial proteases (Alcalase, Flavourzyme, Protamex, and Neutrase). Yields of all the produced WGH were over 50%. Degree of hydrolysis of the WGH produced by Flavourzyme was the highest, while that of the WGH by Neutrase was the lowest. Sequential enzymatic hydrolysis remarkably decreased the fraction with more than 10 kDa and increased the fraction with less than 500 Da. Contents and composition of free amino acids were affected by the enzyme types and hydrolysis conditions. DPPH radical scavenging activity of the WGH significantly increased with hydrolysis time. The WGH produced by Protamex showed lower turbidity, better thermal stability, and higher solubility, suggesting they may be suitable for beverage development.

Wheat gluten hydrolysates and their fractions improve multiple stress tolerance and ethanol fermentation performances of yeast during very high-gravity fermentation

Wheat gluten hydrolysates (WGH) and their fractions (0%, 20% and 40% ethanol fractions) obtained on XAD-16 were used to examine their effects on multiple stress tolerance and ethanol fermentation performances of yeast during very high-gravity (VHG) fermentation. Results showed that WGH and their fractions supplementations increased significantly osmotic/ethanol stress tolerance, growth and vitality of yeast cells. Supplementation with 40% ethanol fraction resulted in 46.54% and 35.01% of increase in cell membrane integrity, and 42.59% and 21.79% of reduction in intracellular ROS level under osmotic and ethanol stress conditions, respectively. Moreover, WGH and their fractions supplementations also increased physiological activity and ethanol yield of yeast cells under VHG fermentation, and 40% ethanol fraction supplementation caused the maximum biomass and ethanol production during VHG fermentation. These results suggested that WGH supplementation was an efficient and convenient method for improvement of yeast cells with enhanced multiple stress tolerance and ethanol fermentation performances.

Extraction and Purification of Protease from Nigella sativa for Its Potential Use in Celiac Disease

Celiac disease (CD) is a disease of the digestive system resulting from the intolerance of autoimmune system against gluten and gliadin proteins in wheat, barley, rye and some varieties of oats. In this work protease was isolated and partially purified from Nigella Sativa using ammonium sulphate, acetone and trichloroacetic acid. The purified protease was evaluated further for its possible use in celiac disease by its ability to hydrolyse both, gluten and gliadin. Data indicate that the highest purity of digestive protease enzymes was obtained by acetone 80% and 0.2M trichloroacetic acid (TCA). The enzyme purified by acetone exhibited the high value of Vmax (166.66 mg/min) and the low value of Km (0.133 mg/ml). When the obtained enzyme was tested to hydrolyse gluten and gliadin at two increasing concentrations of 1 and 2 mg/ml, almost similar efficacy was observed (28.46% and 32.02%; for gluten) and (22.7% and 16.84%; for gliadin), whereas Digestin, a positive control which contain Papain and Sanzyme 3500 and works by helping in the breakdown of proteins, produced higher efficacy at 2 mg/ml to hydrolyse gluten (38.67%) with 1 mg/ml (25.68%) whereas similar results were observed against gliadin at both concentrations (22.06% and 22.5%). This study show the efficacy of protease obtained from Nigella Sativa as a natural and economical source for the hydrolysis of gluten and gliadin, which is considered the primary reason for celiac disease.

Effect of acid deamidation-alcalase hydrolysis induced modification on functional and bitter-masking properties of wheat gluten hydrolysates.

The low solubility of wheat gluten (WG) considerably limits its application. Owing to its high hydrolytic efficiency, alcalase was the protease selected for the enzymatic hydrolysis of WG. The functional properties of WG hydrolysate prepared by alcalase (AHWG) with a hydrolysis degree (DH) of 10% were better than those with DH 5% and DH 15%. The application of AHWG was hindered by its bitterness. To mask the bitterness of AHWG, WG was respectively deamidated with acetic acid, tartaric acid, and citric acid, followed by being hydrolyzed by alcalase to DH 10%. The citric acid deamidation-alcalase hydrolysis WG hydrolysate (CDAH) exhibited the best functional properties. Partial least squares regression analysis results indicated that CDAH exhibited an enhanced bitter-masking property attributable to a high content of umami taste amino acids (glutamic acid and aspartic acid). Thus, CDAH showed the greatest potential as a modified WG product to expand the application of WG.

Wheat gluten hydrolysate potently stimulates peptide-YY secretion and suppresses food intake in rats.

The study was aimed to compare the satiating effect of various protein hydrolysates in rats and examine the underlying mechanism associated with the satiety hormones. Food intake and portal satiety hormone levels were measured in rats. Enteroendocrine cell-lines were employed to study the direct effect of protein hydrolysates on gut hormone secretions. The results showed that oral preload of wheat gluten hydrolysate (WGH) suppressed food intake greater and longer than other hydrolysates. The portal peptide-YY levels in WGH-treated rats at 2 h and 3h were higher than those in control- and lactalbumin hydrolysate (LAH)-treated rats. In a distal enteroendocrine cell model, WGH more potently stimulated glucagon-like peptide-1 secretion than LAH, and the effect was largely enhanced by pepsin/pancreatin digestion of WGH. These results suggest WGH is potent in activating enteroendocrine cells to release satiety hormones leading to the prolonged suppression of food intake.

Potential yeast growth and fermentation promoting activity of wheat gluten hydrolysates and soy protein hydrolysates during high-gravity fermentation

Wheat gluten hydrolysates (WGH) and soy protein hydrolysates (SPH) were used to examine their effects on the growth and fermentation performance of yeast during high-gravity fermentation. Results showed that both the kind and the addition amount of hydrolysates had significant impacts on the biomass, viability, fermentability and ethanol yield of yeast cells. Worts supplemented with WGH and SPH (with the hydrolysis time of 24 h and 12 h, and the addition amount of 1.0% and 0.1%, respectively) increased 5.5% and 12.6% of net cell growth, 0.3% and 1.1% of fermentability, and 19.3% (v/v) and 23.7% (v/v) of ethanol yields, respectively. All these results indicated that both hydrolysates were effective and preferential nitrogen sources for yeast growth and fermentation. The moderate molecular weight distribution, the complexity of nitrogen source and the higher hydrophilic and basic amino acids contents in hydrolysates might be responsible for their specific bioactivity.

Preparation of a Defined Gluten Hydrolysate for Diagnosis and Clinical Investigations of Wheat Hypersensitivities.

Gluten is the trigger for celiac disease (CD), non-celiac gluten/wheat sensitivity (NCGS), and wheat allergy. An oral food challenge is often needed for diagnosis, but there are no standardized gluten challenge materials with known composition available. To fill this gap, two materials, commercially available gluten and a food-grade gluten hydrolysate (pepgluten), were extensively characterized. Pepgluten was prepared from gluten by incubation with a pepsin dietary supplement and acetic acid at 37 °C for 120 min. The components of pepgluten were crude protein (707 mg/g), starch (104 mg/g), water (59 mg/g), fat (47 mg/g), dietary fiber (41 mg/g) and ash (11 mg/g). The protein/peptide fraction of pepgluten (1 g) contained equivalents derived from 369 mg gliadins and 196 mg glutenins, resulting in 565 mg total gluten equivalents, 25 mg albumins/globulins, 22 mg α-amylase/trypsin inhibitors and 48 mg pepsin capsule proteins. The slightly acidic, dough-like smell and bitter taste of pepgluten could be completely camouflaged in multivitamin juice with bitter lemon, grapefruit juice, or vegetable and fruit smoothies. Thus, pepgluten met the criteria for placebo-controlled challenges (active and placebo materials are identical regarding appearance, taste, smell, and texture) and is appropriate as a standard preparation for the oral food challenge and clinical investigations to study wheat hypersensitivities.

Enzymatically Hydrolyzed Wheat Gluten as a Foaming Agent in Food: Incorporation in a Meringue Recipe as a Proof‐of‐Concept

Replacing animal proteins with plant protein sources in the food industry is desirable from an economic and environmental perspective. Enzymatic hydrolysis serves as a tool to improve the foaming properties of water-insoluble wheat gluten proteins. We conclude that wheat gluten hydrolysates can be a valid functional alternative for egg white proteins in meringues, and possibly other food systems.

Identification of food-derived peptides in human blood after ingestion of corn and wheat gluten hydrolysates

Bioactive peptides in the body after ingestion of plant protein hydrolysates have been speculated but not identified. We aimed to establish an approach to identify small amounts of food-derived peptides in humans after ingestion of non-extracellular matrix protein hydrolysates. Corn and wheat gluten hydrolysates were digested using pancreatin and leucine aminopeptidase; the resultant peptides were identified via size-exclusion chromatography and reverse-phase HPLC-tandem mass spectrometry (MS/MS). Structures of indigestible peptides were confirmed via LC-MS/MS in multi-reaction monitoring mode. All indigestible peptides in the exopeptidase digest were diprolyl and di- and tripyroglutamyl peptides. Blood collected from healthy volunteers (n = 4) before and after ingestion of 9 g of the hydrolysates was assessed for the indigestible peptides via LC-MS/MS. Six peptides (Pro-Ala, Pro-Gly, Pro-Gln, pyroGlu-Pro, pyroGlu-Leu-Pro, and pyroGlu-Gln-Pro) significantly increased in human plasma up to 10–100 nM compared to the baseline. This may hence be a powerful tool for identifying foodderived peptides in blood.

Nanomagnetic wheat gluten hydrolysates a new carrier for nanoimmobilization of inulinase

Functionalized magnetic nanoparticles are effective enzyme carriers since they have several advantages such as easily separation under external magnetic fields as well as reusability of enzymes. In this study, it was tried to prepare a composite nanoparticle with Fe3O4 nanoparticles and wheat gluten hydrolysate nanoparticles for utilizing in biocatalyst production. Firstly, Fe3O4 nanoparticles were synthesized by chemical co-precipitation. Since these nanoparticles are easily aggregated, surface coating procedure was used. These nanoparticles were coated by wheat gluten hydrolysates (WGHs) using glutaraldehyde as crosslinking agent. WGHs were prepared by enzymatic hydrolysis of wheat gluten by endopeptidases with degree of hydrolysis (24.3%) and then, by desolvation method using ethanol as a desolvating agent. FE-SEM images and DLS analysis showed that Fe3O4 nanoparticles have spherical morphology with 30–40 nm in diameter. Also, wheat gluten hydrolysates were ranged 100–150 nm and their zeta potential was around −29 mV. It should be noted that the size of nanocomposite particles were about 120–160 nm. Inulinase was covalently and successfully immobilized through gluteraldehyde on Fe3O4@WGH nanoparticles. Moreover, The results implied that, the enzyme was loaded up to 75% onto functionalized nanoparticles.